The type 3 serotonin (5-HT3) receptor is a ligand-gated ion channel. Pharmacological studies had shown that the 5-HT3-receptor facilitates dopamine release in the nucleus accumbens. The 5-HT3-receptor antagonists reduce extracellular DA induced by 5-HT3-receptor agonists, drugs of abuse and by the direct stimulation of dopaminergic neurons. The source of the 5-HT3-receptor that regulates DA release is unknown. Although pharmacological studies suggest that the 5-HT3-receptor may be present in the nucleus accumbens and ventral tegmental area, prior anatomical studies have revealed few or no 5-HT3-receptor binding sites in these regions. By using in situ hybridization histochemistry, we detected expression of the functional 5HT3A subunit, but not expression of the 5HT3B subunit in the rat midbrain. The 5HT3A subunit was found in the ventral tegmental area, parabrachial pigmented nucleus, substancia nigra pars compacta, substantia nigra pars lateralis, prerubral field, medial and supra mammillary nucleus, and interpeduncular nucleus. To determine the cellular phenotype of 5HT3A expressing neurons, we used double-labeling techniques and determined that many dopaminergic cells express the 5HT3A subunit in the substantia nigra, ventral tegmental area and interpeduncular nucleus. In addition, the 5HT3A subunit was found to be expressed in non-dopaminergic neurons located in the substantia nigra pars reticulata, substantia nigra pars lateralis and ventral tegmental area. The localization of 5HT3A subunit transcripts in dopaminergic neurons suggests that serotonin, through 5-HT3-receptors present in the midbrain, may regulate the release of dopamine. In addition, expression of the 5HT3A subunit in midbrain non-dopaminergic neurons suggests that serotonin might modulate dopaminergic neuronal activity via 5-HT3-receptors distributed in local GABAergic neurons. However, dopamine neurotransmission may be also regulated by 5-HT3-receptors distributed within areas innervating the striatum and midbrain.